Alleviation of allelopathic stress of benzoic acid by indole acetic acid in Solanum lycopersicum

Scientia Horticulturae 192 (2015) 211–217

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Alleviation of allelopathic stress of benzoic acid by indole acetic acid in Solanum lycopersicum Sunaina, N.B. Singh ∗ Plant Physiology Laboratory, Department of Botany, University of Allahabad, Allahabad 211002, India

a r t i c l e

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Article history: Received 21 February 2015 Received in revised form 5 June 2015 Accepted 7 June 2015 Keywords: Antioxidant system Benzoic acid Electrolyte leakage Indole-3-acetic acid Lipid peroxidation Oxidative stress

a b s t r a c t The present study investigates the alleviation of allelochemical stress induced by benzoic acid (BA), an autotoxic compound, by exogenous indole acetic acid (IAA) in tomato seedlings. The experiment was conducted in hydroponic culture in glass house conditions. BA was applied 0.5 and 1.0 mM concentrations with and without IAA (1.0 mM). Root and shoot length, fresh and dry weight, pigment, protein, sugar content and nitrate reductase activity decreased in the seedlings treated with BA, while they increased in BA combined with IAA. Lipid peroxidation, electrolyte leakage and proline level enhanced in the seedlings under BA stress, but these parameters decreased in combined BA + IAA treatment. IAA protected the seedlings against oxidative stress caused by reactive oxygen species (ROS). IAA buttressed defense system of tomato plant against BA toxicity which was evident from increased activities of antioxidant enzymes. IAA has potential to enhance tolerance to stress in the tomato seedlings under allelochemical toxicity. © 2015 Published by Elsevier B.V.

1. Introduction Autotoxicity is a common phenomenon in the natural and agro-ecosystems (Liu et al., 2007; Zhang et al., 2010). A process in which a plant species or its decomposing plant parts release phytotoxins into the surroundings to inhibit the growth and development of the same species is generally known as autotoxicity or autointoxication (Miller, 1996; Sannigrahi and Chakrabortty, 2005; Cruz-Ortega et al., 2008). Autotoxicity prevalent in monocropping system decreases production and yield equally in agriculture and forestry (Chou and Lin, 1976; Baziramakenga et al., 1995; Cao and Luo, 1996; Batish et al., 2001; Bonanomi et al., 2007; Zhang et al., 2010). The various physiological processes of economical important crops are adversely affected in monoculture due to autotoxity. In natural conditions plants are rarely subjected to a single stress factor, rather a group of factors. Allelochemicals are low molecular weight secondary metabolites which interfere with various metabolic processes in plants. Allelochemicals influence a number of physiological processes (Blum, 1995; Inderjit and Duke, 2003; Singh et al., 2010). Tomato is major commercial vegetable crop grown worldwide. Continuous cropping of tomato for long period in the same field may lead to soil sickness (Zheng et al., 2004; Enping et al., 2015).

∗ Corresponding author. E-mail address: [email protected] (N.B. Singh). http://dx.doi.org/10.1016/j.scienta.2015.06.013 0304-4238/© 2015 Published by Elsevier B.V.

Accumulation of allelopathins in monocropping decrease plant growth and yield (Yu et al., 1993; Yu and Matsui, 1997). Autotoxicity related soil problems in tomato are commonly reported (Yu and Matsui, 1997; Wu et al., 1997). Aqueous extract of tomato plant and hydroponic medium from tomato culture are autotoxic and decreased plant height and biomass (Zhou et al., 1997; Singh et al., 2008). Exudates from different plant parts of tomato are allelopathic to lettuce (Kim and Kil, 1989). The presence of di-isooctyl phthalate, di-iso-butyl phthalate, tannic acid and salicylic acid in tomato plants are reported by Zhou et al. (1998). Allelochemicals are released into soil through leaching, volatilization, root exudation, microbial decomposition and microbial phytotoxins. Allelochemicals released as a mixture of compounds. The effects of individual allelochemicals are often different from their mixture. Tomato leaf extract has inhibitory effects on growth and biomass of the seedlings (Bonanomi, 2007). Roots are in close contact with allelopathic compounds released in growth medium. Phenolics compounds viz. salicylic acid, vanillic acid and genlisic acid, benzoic acid, palmitic acid, sinapic acid, para-hydroxybenzoic acid, ferulic acid,caffeic acid and synaptic acid are reported in tomato plants (Yu and Matsui, 1997; Mizutani, 1984). Benzoic acid (BA) is one of the common secondary metabolites with allelopathic potential which affects growth and metabolism of plants. Soil sickness caused by autotoxicity has significant impact on tomato productivity. BA derivatives extracted from soil suppress growth and development of plants (Vaughan and Ord, 1991). Allelochemicals cause oxidative stress which impose injurious effects

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on membrane stability via lipid peroxidation (Lin et al., 2000), DNA damage, protein oxidation and ultimately cell death (Cruz-Ortega et al., 2002). Plants develop several defence mechanisms to cope with oxidative stress (Singh et al., 2010). Production of reactive oxygen species (ROS) scavengers is an important device to increase tolerance against oxidative stress (Sairam et al., 1998). Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and guaiacol peroxidase (GPX) are antioxidant enzymes which detoxify ROS (Rubio et al., 2002; Ünyayar et al., 2005) and protect crop plants in oxidative stress. Yadav and Singh (2013) reported that BA at 0.5, 1.0 and 1.5 mM concentrations exhibited toxic effects on crop plants. IAA was used to ameliorate environmental stresses like salt stress (Torres-Garcia et al., 2009; Javid et al., 2011; Kaya et al., 2013), water stress (Torres-Garcia et al., 2009), stress tolerance and adaptation (Egamberdieva, 2009), metal stress (Gangwar et al., 2011). IAA is the only natural auxin responsible for cell elongation and lateral root formation which increased absorption and accumulation of minerals causing increased growth of crops plants (Egamberdieva, 2009). Exogenous IAA can promote the plant growth and alleviate the harmful effects of biotic and abiotic stresses in plants (Gangwar et al., 2011; Javid et al., 2011; Kaya et al., 2013). Stresses declined endogenous level of IAA (Wang et al., 2001). IAA actively participates in mobilization and accumulation of carbohydrates in seeds (Kato and Takeda, 1993). Exogenous auxin alleviates the adverse effects of stress and thus improves germination, growth, development and yields and quality of crops (Khan et al., 2004; Egamberdieva, 2009). The two concentrations viz. 0.5 and 1.0 mM of BA were selected as a range of low doses with low toxic effects on the basis of lethal dose (LD50) to record the efficiency of IAA to mitigate the allelopathic stress caused by the allelochemical. It is reported that 0.5, 1.0 and 1.5 mM concentrations of BA exhibited toxic effects on crop plants (Yadav and Singh, 2013). Phytohormones are used to ameliorate various environmental stresses (Unal, 2013). However, the role of phytohormones under allelopathic stress is not well studied. The aim of the present study was to explore the interactive effect of IAA and BA on growth and metabolism of tomato crop grown under allelopathic stress in hydroponic culture. 2. Materials and methods 2.1. Seeds and chemicals Seeds of tomato (Solanum lycopersicum) var. Pusa ruby were procured from seed agency in Allahabad, India. Benzoic acid (BA) was purchased from Loba Chemie Pvt., Ltd., Mumbai. Indole-3 acetic acid (IAA) (molecular weight: 175.19 g/mol) was purchased from CDH. 2.2. Hydroponic culture The seeds were sown in October, 2014, in nursery beds (1 mX1 m) in the garden of the Department of Botany, University of Allahabad, Allahabad, India. The seed bed was irrigated as and when required. Twenty-one-days old seedlings of uniform size were uprooted and washed under tap water to remove soil adhering on roots. The seedlings were transferred in transparent plastic boxes (23 × 17 × 9 cm) filled with 2 L (L) half strength Hoagland solution (Hoagland and Arnon, 1950). Six seedlings in each box were planted at equal distance. One week after the establishment of the seedlings in hydroponic culture, the boxes were divided into five sets of three each. In one set, Hoagland solution was replaced by fresh Hoagland solution (2 L) and was taken as control. In the second and third sets, Hoagland solution was replaced by fresh Hoagland

solution (2 L) each containing two different concentrations viz. 0.5 and 1.0 mM of BA. In the fourth and fifth sets, Hoagland solution was replaced by Hoagland solution (2 L) each containing BA and IAA viz. BA1 (0.5 mM)+IAA (1.0 mM) and BA2 (1.0 mM)+IAA (1.0 mM). Stock solution (1.0 mM i.e. BA2 ) of BA was prepared by dissolving requisite amount in 100 mL double distilled water (DDW). The stock solution (1.0 mM) was further diluted with DDW to get 0.5 mM (BA1 ) concentration of BA. Requisite amount of IAA was dissolved in 100 mL DDW to obtained 1.0 mM concentration of IAA. The experiment was conducted in three replicates. The boxes were aerated for 12 h a day with the help of bubblers. The boxes were covered with black paper to avoid the algal growth in the medium. The sampling was done after one week of the treatment. The first fully expanded leaves of the seedlings were sampled for biochemical analyses. Root and shoot length and fresh and dry weight of the seedlings were recorded. 2.3. Pigment and protein contents The leaves (10 mg) were homogenized with 10 mL of 80% acetone. Chlorophylls and carotenoids were extracted and quantified following the method of Lichtenthaler (1987). Protein content was determined according to the method of Lowry et al. (1951). The amount of protein was calculated with reference to the standard curve obtained from bovine serum albumin. 2.4. Sugar content The estimation of total soluble sugars (TSS) was done following Hedge and Hofreiter (1962). About 0.1 g fresh leaf was homogenized in 5 mL 95% (v/v) ethanol. After centrifugation, 1 mL supernatant was mixed with 4 mL anthrone reagent and heated on boiling water bath for 10 min. Absorbance was recorded at 620 nm after cooling. The amount of sugar was determined by the standard curve prepared from glucose. 2.5. Nitrate reductase activity Nitrate reductase (NR) activity was measured following the procedure of Jaworski (1971). Fresh leaf tissue (0.25 g) was incubated in 4.5 mL medium which contained 100 mM phosphate buffer (pH 7.5), 3% (w/v) KNO3 and 3 N HCl and 0.02% (w/v) N(1-naphthyl)ethylene diamine dihydrochloride. The absorbance was recorded at 540 nm. NR activity was measured with standard curve prepared from NaNO2 and expressed as nmol NO2 mg protein−1 h−1 . 2.6. Membrane leakage Membrane integrity was assessed in terms of electrolyte leakage. Fresh leaf samples (0.1 g) were placed in a vial containing 10 mL of deionized water and allowed to stand in dark for 24 h at room temperature. Electrical conductivity (EC1 ) of the bathing solution was measured at the end of incubation period. The tissue with bathing solution was then heated in water bath at 95 ◦ C for 20 min and the electrical conductivity (EC2 ) was again measured after cooling. Electrolyte leakage was calculated as percentage of EC1 /EC2 . 2.7. Lipid peroxidation Lipid peroxidation was measured as the amount of malondialdehyde (MDA) determined by thiobarbituric acid reactive substance as described by Heath and Packer (1968). Fresh leaf (0.2 g) was ground in 0.1 w/v trichloroacetic acid (TCA) and centrifuged at 10,000g for 10 min. One milliliter supernatant was mixed with 4 mL

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of 0.5% thiobarbituric acid prepared in 20% (w/v) TCA. The mixture was then heated at 95 ◦ C for 30 min and again centrifuged after cooling. The absorbance of the supernatant was recorded at 532 nm and corrected by subtracting the non-specific absorbance at 600 nm. The MDA concentration was calculated using the extinction coefficient of 155 mM−1 cm−1 and expressed as nmol g−1 FW. 2.8. Free proline Extraction and determination of proline were done following Bates et al. (1973). Leaf samples were extracted with 3% sulphosalicylic acid. Aliquot was treated with acid ninhydrin and acetic acid, boiled for 1 h at 100 ◦ C. The reaction mixture was extracted with 4 mL of toluene. Absorbance of chromophore containing toluene was determined at 520 nm. Proline content was expressed as ␮mol g−1 fresh weight. 2.9. Extraction and assay of antioxidant enzymes Fresh leaves (0.25 g) were homogenized with 0.1 M sodium phosphate buffer containing 1% (w/v) polyvinyl pyrrolidone (pH 7.0) in a pre-cooled mortar and pestle. The extract was centrifuged at 48 ◦ C at 14,000g for 30 min in cooling centrifuge (Remi instruments C 24). The supernatant was used for the assay of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and guaiacol peroxidase (POX) activities. 2.10. Assay of SOD activities The activity of SOD (EC 1.15.1.1) was estimated by the nitroblue tetrazolium (NBT) photochemical assay following Beyer and Fridovich (1987). The reaction mixture (4 mL) consisted of 20 mM methionine, 0.15 mM ethylene diamine-tetra acetic acid (EDTA), 0.12 mM NBT and 0.5 mL supernatant. The test tubes were exposed to fluorescent lamp for 30 min and identical unilluminated assay mixture served as blank. One unit of enzyme was measured as the amount of enzyme which caused 50% inhibition of NBT reduction. 2.11. Assay of CAT activities CAT (EC1.11.1.6) activity was assayed following the method by Cakmak and Marschner (1992). Assay mixture (2 mL) contained 25 mM potassium phosphate buffer (pH 7.0), 10 mM H2 O2 , and 0.5 mL enzyme extract. The rate of H2 O2 decomposition for 1 min was monitored at 240 nm and calculated using extinction coefficient of 39.4 mM−1 cm−1 and expressed as enzyme unit mg−1 protein. One unit of CAT was determined as the amount of enzyme required to oxidize 1 mM H2 O2 min−1 . 2.12. Assay of APX activities APX (EC1.11.1.11) activities were assayed following Nakano and Asada (1981). Assay mixture (2 mL) contained 25 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.25 mM ascorbate, 1.0 mM H2 O2 , and 0.2 mL enzyme extract. H2 O2 was the last component to be added. The absorbance was recorded for 1 min at 290 nm (extinction coefficient of 2.8 mM−1 cm−1 ). Enzyme specific activity was measured as enzyme unit per one milligram protein as the amount of enzyme required to oxidize 1 mM H2 O2 min−1 .

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absorbance due to oxidation of guaiacol was monitored at 470 nm. The enzyme activities were measured using extinction coefficient of 26.6 mM−1 cm−1 and expressed as enzyme unit per mg protein. 2.14. Statistical analysis Treatments were arranged in a randomized block design with three replications. Data were statistically analyzed using analysis of variance (ANOVA) by using SPSS software (Ver. 10; SPSS Inc., Chicago, IL, USA). Appropriate standard error of means (±SEM) was calculated for presentation with tables and graphs. The treatment means were analyzed by Duncan’s multiple range test (DMRT) at P<0.05. 3. Results 3.1. Growth of seedlings Allelopathic stress caused by BA adversely affected the seedlings growth as compared with control. Root (RL) and shoot (SL) length of seedlings significantly decreased under the influence of BA stress. Maximum RL and SL recorded under the control group decreased in BA and corresponded to concentrations of BA. The combined treatments (BA + IAA) improved both root and shoot growth in comparison to that of BA treatments. FW and DW of seedlings significantly P<0.05) decreased in both concentrations of BA treatment in comparison to control with maximum decrease in BA2 . IAA enhanced RL, SL, FW and DW of the seedlings under allelopathic stress (Table 1). 3.2. Pigment content Allelochemicals caused decrease in pigment content. Maximum amount of chlorophylls and carotenoids in control group significantly decreased in dose dependent manner under allelochemical stress. BA1 and BA2 caused maximum 37.93 and 46.35% decrease in total chlorophyll and 59.83 and 68.51% decrease in carotenoids content, respectively, as compared with control, while total chlorophyll content significantly improved to 28.84 and 29.74% and carotenoids 38.48 and 24.45% in BA1 + IAA and BA2 + IAA treatments, respectively as compared with BA treatments (Table 2). 3.3. Sugar and protein content Total soluble sugar (TSS) of the seedlings was negatively affected in BA treatments. Significant reduction in sugar content was recorded in tomato seedlings exposed to allelochemicals with maximum 27.77 and 36.66% in BA1 and BA2 , respectively, as compared with control. TSS remarkably 19.75 and 22.97% increased in the combined treatments in BA1 + IAA and BA2 + IAA, respectively, as compared with that of BA (Table 3). Protein content decreased in the seedlings treated with BA. The inhibition of protein content was concentration dependent. Significant 32.10 and 45.05% inhibition of protein content was recorded in the seedlings treated with BA1 and BA2 , respectively, as compared with control. Significant 28.22 and 33.40% stimulation of protein content was recorded in BA1 + IAA and BA2 + IAA treatments, respectively, as compared with that of BA treatments (Table 3).

2.13. Assay of guaiacol POX activities 3.4. Nitrate reductase activity Guaiacol POX (EC 1.11.1.7) activities were assayed following Hemeda and Klein (1990). The reaction mixture (2 mL) contained 25 mM phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.05% (v/v) guaiacol, 1.0 mM H2 O2 , and 0.2 mL of enzyme extract. The increase in

The data showed that nitrate reductase (NR) activity declined significantly (P < 0.05) in the leaves of tomato seedlings under the influence of BA. A concentration dependent decrease in NR activity

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Table 1 Effect of benzoic acid on shoot length, root length, fresh weight and dry weight of tomato seedlings grown in hydroponic culture with or without IAA. Treatment C BA1 BA1 + IAA BA2 BA2 + IAA

Shoot length (cm)

Root length (cm)

24.70 ± 0.315a 19.93 ± 0.924 b 21.26 ± 0.284ab 17.43 ± 0.296b 20.16 ± 0.287b

10.5 6.2 8.6 5.4 7.5

± ± ± ± ±

Fresh weight (g/plant)

0.015a 0.493cd 0.971b 0.305d 0.360bc

4.87 2.69 2.86 2.34 2.57

± ± ± ± ±

Dry weight (g/plant)

0.437a 0.019b 0.109b 0.003b 0.151b

0.925 0.626 0.879 0.543 0.617

± ± ± ± ±

0.033a 0.016c 0.005a 0.003d 0.008b

Mean ± SE values followed by same letters within each column are not significantly different at 0.05 (ANOVA and Duncan’s multiple range test), n = 3. IAA (1.0 mM), BA1 = 0.5 and BA2 = 1.0 mM concentrations of benzoic acid. Table 2 Effect of benzoic acid on pigment contents of tomato seedlings grown in hydroponic culture with or without IAA. Treatment

Pigment (mg/g FW) Chlorophyll a

C BA1 BA1 + IAA BA2 BA2 + IAA

1.225 0.915 1.114 0.670 1.029

± ± ± ± ±

Chlorophyll b

0.024a 0.025c 0.042ab 0.025d 0.004b

0.818 0.425 0.638 0.353 0.490

± ± ± ± ±

Total chlorophyll

0.012a 0.008d 0.060b 0.003cd 0.021c

2.043 1.268 1.782 1.096 1.560

± ± ± ± ±

Carotenoides

0.036a 0.029d 0.017b 0.033e 0.017c

0.991 0.398 0.647 0.312 0.413

± ± ± ± ±

0.001a 0.001d 0.001b 0.003e 0.001c

Mean ± SE values followed by same letters within each column are not significantly different at 0.05 (ANOVA and Duncan’s multiple range test), n = 3. IAA (1.0 mM), BA1 = 0.5 and BA2 = 1.0 mM concentrations of benzoic acid. Table 3 Effect of benzoic acid on sugar and protein content and NR activity of tomato seedlings grown in hydroponic culture with or without IAA. Treatment

C BA1 BA1 + IAA BA2 BA2 + IAA

Sugar (mg/g FW) 45.0 32.5 40.5 28.5 37.0

± ± ± ± ±

2.886a 0.288cd 0.288ab 0.866d 1.154bc

Protein (mg/g FW) 27.72 18.82 26.22 15.23 22.87

± ± ± ± ±

NR activity (␮mol NO2 g−1 FW h−1 )

0.967a 0.216c 0.591a 0.066d 1.169b

23.25 17.5 20.62 11.87 19.37

± ± ± ± ±

0.144a 0.721c 0.360b 1.082d 0.360bc

Mean ± SE values followed by same letters within each column are not significantly different at 0.05 (ANOVA and Duncan’s multiple range test), n = 3. IAA (1.0 mM), BA1 = 0.5 and BA2 = 1.0 mM concentrations of benzoic acid.

was recorded. The maximum NR activity was recorded in control decreased to minimum in BA2 . Maximum 24.73 and 48.94% decrease in NR activity was recorded in leaves of seedlings of BA1 and BA2 treatments, respectively as compared with control. Maximum 15.13 and 38.72% enhancement in NR activity was observed in BA1 + IAA and BA2 + IAA treatments, respectively, over BA (Table 3).

d

b e a

c

3.5. Lipid peroxidation and electrolyte leakage Lipid peroxidation (LP) and electrolyte leakage (EL) increased in the seedlings exposed to BA. LP was measured in terms of MDA content. Lowest amount of MDA and EL was recorded in the seedlings under control. A gradual increase in MDA level and EL was recorded in both treatments of BA. MDA content and EL decreased in combined (BA + IAA) as compared with treatments with respective concentration of BA. Exogenous IAA significantly (P < 0.05) maintained level the LP and EL in the seedlings exposed to allelochemical to overcome the stress (Figs. 1 and 2).

3.6. Proline content A significant (P < 0.05) accumulation of proline was recorded in leaves of the BA stressed seedlings as compared with control. Leaf proline was not affected in combined treatments. Significant decrease in proline content was recorded in the seedlings exposed to combined BA + IAA treatment as compared with that of BA treatments (Fig. 2).

Fig. 1. Effect of benzoic acid on electrolyte leakage of tomato seedlings grown in hydroponic culture with or without IAA. Mean ± SE values followed by same letters within each column are not significantly different at 0.05 (ANOVA and Duncan’s multiple range test), n = 3. IAA (1.0 mM), BA1 = 0.5 and BA2 = 1.0 mM concentrations of benzoic acid.

3.7. Activities of antioxidant enzymes Our results indicated that exposure of tomato seedlings to BA alone and together with IAA significantly affected the antioxidant machinery. The antioxidant enzymes viz. SOD, CAT, APX and

Sunaina, N.B. Singh / Scientia Horticulturae 192 (2015) 211–217

b

bc

c

b

c c ab

b

a a

Fig. 2. Effect of benzoic acid on lipid peroxidation and proline content of tomato seedlings grown in hydroponic culture with or without IAA. Mean ± SE values followed by same letters within each column are not significantly different at 0.05 (ANOVA and Duncan’s multiple range test), n = 3. IAA (1.0 mM), BA1 = 0.5 and BA2 = 1.0 mM concentrations of benzoic acid.

e c d ab bc

b

a

b

b

b

a c

c

d c

bc

ab ab a

bc

Fig. 3. Effect of benzoic acid on antioxidant enzymes activity of tomato seedlings grown in hydroponic culture with or without IAA. Mean ± SE values followed by same letters within each column are not significantly different at 0.05 (ANOVA and Duncan’s multiple range test), n = 3. IAA (1.0 mM), BA1 = 0.5 and BA2 = 1.0 mM concentrations of benzoic acid.

GPX activities showed different responses when seedlings were exposed to single and combined treatments of BA and IAA (Fig. 3). The SOD activity slightly increased in response to allelopathic stress. In combined BA + IAA treatments activity of SOD significantly (P < 0.05) enhanced as compared with that of BA treatments. CAT activity slightly increased in BA treatments as compared with control. It was noticed that exposure of tomato seedlings to BA + IAA highly increased the CAT activity. IAA exhibited significant effect on CAT activity. APX and GPX activities were significantly affected by allelopathic stress. Application of IAA together with BA treatments stimulated APX and GPX activities as compared to BA treatments. 4. Discussion Plants compete for natural resources like space, water, light and essential nutrients. They are also exposed to allelopathic stress in natural and agro ecosystems. The plants have allelopathic interactions among themselves. Biomass is an index of plant growth. Decrease in seedlings growth and reduction in biomass evinced

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the autotoxic nature of the tomato plants. Various strategies are in practice to neutralize the adverse effects of allelopathic stress on commercial crop plants for sustainable yield. Exogenous application of plant growth regulators and osmoprotectants may be an economical move to alleviate the detrimental effects of stresses on crop plants (Ashraf et al., 2008; Kaya et al., 2013). IAA improves the growth and metabolism of plants exposed environmental stresses (Iqbal and Ashraf, 2007; Guru Devi et al., 2012; Kaya et al., 2013). Allelochemicals present in tomato seedlings result in autointoxication (Sannigrahi and Chakrabortty, 2005; Zhang et al., 2009 Singh et al., 2010). Decrease in root and shoot growth may be due to inhibition of the cell division, elongation and expansion of cells and impairment of metabolic activities in the seedlings under allelopathic stress (Sheltel and Balke, 1983). Roots are in direct contact with BA. Inhibition of root growth decreased shoot growth and reduced photosynthetic area. Allelochemical inhibits the electrical conductivity and nutrient uptake by roots which result in growth inhibition (Blum, 1995). Reduced plant growth may be a strategy of plants in allelopathic stress to save energy when absorptive surface and photosynthetic area and pigments decreased. Potential growth regulators are considered to be effective means to mitigate the oxidative stress induced by allelochemicals to improve the growth of crop plants. Exposure of plants to IAA causes loosening and stretching of cell walls which result in cell expansion and elongation. Expansion and elongation of cells increased seedling growth (Thapar and Singh, 2005). The stimulated plant growth may be due to increase in cell size and cell division (Chaudhary and Khan, 2007; Wang et al., 2000) and cell enlargement (Abdoli et al., 2013). It is evident from our results that IAA regulates the action of BA and expresses positive effects on growth of the tomato seedlings under allelochemical stress. Studies on role of IAA in growth regulation in seedlings reveal the positive effect of IAA in adverse condition (Azooz et al., 2004; Chaudhary and Khan, 2007). Decrease in photosynthetic pigments in allelopathic stress caused low photosynthetic rate and low accumulation of photosynthates (Rice, 1984). Reduced cell division, elongation and expansion of cells (Sheltel and Balke, 1983) inhibit water absorption and ion uptake (Einhellig, 1995; Janovicek et al., 1997). These alternations might have resulted in impaired metabolic activities which arrested plant growth under allelochemical stress. The allelochemical stress may regulate the synthesis and activities of growth hormones such as gibberellins and IAA (Tomaszewski and Thimann, 1966) which decreased plant growth. Exogenous application of IAA may offset the effect of BA. IAA increased chlorophyll content (Kaya et al., 2010; Bideshki et al., 2013) and photosynthetic area which influenced photosynthetic rate and accumulation of photoassimilate in stressed plants (Naeem et al., 2004). Our findings are in agreement with auxin mediated increase in growth of onion (Amin et al., 2007) and pea (Amal et al., 2009). BA treatment influenced chlorophyll biosynthesis and photosynthetic rate and ultimately decreased protein contents (Yadav and Singh, 2013). Decreased protein content may be due to the inhibition of protein biosynthesis or proteolysis under the influence of allelochemical (Singh et al., 2008) or oxidative modification/or decrease of protein content (Fazeli et al., 2007; Møller et al., 2007). The exogenous IAA improves the protein content and promotes growth of the seedlings (Singh and Rathore, 1998). IAA stimulates cell division and cell expansion and elongation of cells and influences synthesis and transport of sugars to the sink which are responsible for the growth in BA stress (Ritenour et al., 1996; Awan et al., 1999; Bhatia and Singh, 2002; Javid et al., 2011). Increased amount of sugar and protein content under the influence of IAA may protect the cells against harmful effect of allelochemical (Bangerth et al., 1985; Javid et al., 2011).

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IAA and BA have antagonistic effects. Nitrate reductase (NR) is a substrate induced enzyme (Crawford and Guo, 2005). BA may inhibit the absorption of nitrate by roots and its transport to leaves (Abd-El Baki et al., 2000). Inhibition of photosynthetic rate and decreased foliar nitrate may affect the NR activity. IAA stimulates NO3 − absorption and assimilation (Kaiser et al., 1993). Increased NR activity in plants exposed to IAA may be due to improved photosynthetic systems and NO3 − uptake by the roots from the soil. IAA increases membrane permeability and influences absorption of NO3 − . Electrolyte leakage is a strong indicator of membrane damage in plants subjected to allelopathic stress (Farkhondeh et al., 2012; Mansour, 2012). Membrane damage and lipid peroxidation are common symptoms of allelochemical stress (Singh et al., 2006) which alter permeability and flux across the plasma membrane. Increased MDA level in response to allelopathic stress reflects the oxidative stress in the tomato seedlings due to free radicals (Nürnberger et al., 1994; Zhang et al., 2010). MDA accumulation is more prominent during allelopathic stress caused by BA. Lipid molecules are much sensitive to oxidation caused by ROS generated under BA stress (Baziramakenga et al., 1995). Lipid peroxidation results in the formation of lipid radicals i.e. lipid peroxides which is an indicator of severe oxidative stress (El-Tayeb, 2005). IAA significantly decreases the electrolyte leakage and maintains membrane integrity in plants exposed to BA stress (Kaya et al., 2013). BA induced electrolyte leakage and decreased pigment contents evinced strong relationship between the two physiological parameters (Chen et al., 1991; Kaya et al., 2001). Reduced pigment content and altered membrane permeability in plants under stress lead to leaf senescence and decreased photosynthetic rate (Niu et al., 1995; De Araújo et al., 2006; Kaya et al., 2013). Accumulation of proline is indicator of BA stress (Ruiz et al., 2002; Singh et al., 2010). Proline is a common compatible solute that accumulates in plants under stress condition. Proline as antioxidant which scavenges free-radicals and protects cell-organelles and enzymatic system (Ruiz, 2002; Okuma, 2004). Significant decrease in free proline level in stressed plants reflects the mitigating role played by IAA (Kaya et al., 2013). Allelochemical stress elevates the activities of antioxidant enzymes viz. SOD, CAT, APX, and GPX in the tomato seedlings as compared with control. Increased activities of antioxidant enzymes reflect the induction of ROS and oxidative damage caused by the allelochemical in the seedlings. Antioxidant enzymes play major role in defense mechanisms of plant. Detoxification of superoxide radical (O− ) to hydrogen peroxide is catalyzed by SOD, a metalloenzyme (Apel and Hirt, 2004). Hence SOD blocks the damage caused by superoxide radicals to cell. Catalase and peroxidase activities increased within cells under stress condition (Niakan et al., 2008; Niakan and Mazandrani, 2009). APX and GPX activities significantly elevate in seedlings treated BA treatment. Catalase in peroxisomes removes the excessive hydrogen peroxide produced during photorespiration and superoxide dismutase activities (Bailly et al., 2002). Peroxidases also scavenge the hydrogen peroxide and protect the cell organelles (Apel and Hirt, 2004). Oxidative stress regulates plant growth. The degree of oxidative stress is the result of equilibrium between the ROS produced and activities of antioxidant enzymes induced. Any imbalance between the two results harmful effect on plants. IAA further stimulates the activities of enzymes of the antioxidative defense system (Kaya et al., 2010) over BA stress to counter the toxic effects of ROS. IAA seems to activate and/or induce the biosynthesis of antioxidant enzymes (Synkova et al., 2004; Tognetti et al., 2012). IAA thus plays an important role in the regulation of BA stress tolerance (Kaya et al., 2009). IAA buttresses the defense system of tomato seedlings.

5. Conclusion The present study reveals that IAA stimulates the biosynthesis of pigments, protein and sugar contents and nitrate assimilation and prevents the electrolyte leakage, lipid peroxidation and proline accumulation due to BA stress. Application of IAA induced protection against allelopathic stress caused by BA. An antagonism exists between BA and IAA which may used to alleviate harmful effects of the allelochemical. IAA as growth regulator may protect crop plants from autointoxication which is one of the major problems in monocropping. This may help improve growth and yields of commercial crops grown worldwide. Acknowledgments The authors are thankful to University grants commission, New Delhi, India for the financial assistance to Sunaina. References Abd-El Baki, G.K., Siefritz, F., Man, H.M., Weiner, H., Kaldenhoff, R., Kaiser, W.M., 2000. Nitrate reductase in Zea mays L. under salinity. Plant Cell. Environ. 23, 515–521. Amal, M., Shraiy, E., Hegazi, A.M., 2009. Effect of acetylsalicylic acid, indole-3-butyric acid and gibberellic acid on plant growth and yield of pea (Pisum sativum L.). Basic Appl. Sci. 3 (4), 3514–3523. Amin, A.A., Rashad, M.E., El-Abagy, H.M.H., 2007. Physiological effect of indole-3-butric acid and salicylic acid on growth, yield and chemical constituents of onion plants. Sci. Res. 3 (11), 1554–1563. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress and signal transduction. Annu. Rev. Plant Biol. 55, 373–399. Ashraf, M., Athar, H.R., Harris, P.J.C., Kwon, T.R., 2008. Some prospective strategies for improving crop salt tolerance. Adv. Agron. 97, 45–110. Bailly, C., Bogatek-Leszczynska, R., Côme, D., Corbineau, F., 2002. Changes in activities of antioxidant enzymes and lipoxygenase during growth of sunflower from seeds of different vigour. Seed Sci. Res. 12, 47–55. Bates, L.S., Walderen, R.D., Taere, I.D., 1973. Rapid determination of free proline for water stress studies. Plant Soil 39, 205–207. Batish, D.R., Singh, H.P., Kohli, R.K., Kaur, S., 2001. Crop allelopathy and its role in ecological agriculture. J. Crop Prod. 4, 121–162. Baziramakenga, R., Leroux, G.D., Simard, R.R., 1995. Effects of benzoic and cinnamic acids on membrane permeability of soybean roots. J. Chem. Ecol. 21, 1271–1285. Beyer, W.F., Fridovich, I., 1987. Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Anal. Biochem. 161, 559–566. Bideshki, A., Arvin, M.J., Darini, M., 2013. Interactive effects of indole-3-butyric acid (IBA) and salicylic acid (SA) on growth parameters, bulb yield and allicin contents of garlic (Allium sativum) under drought stress in field. Int. J. Agron. Plant Prod. 4 (2), 271–279. Blum, U., 1995. The value of model plant-microbe-soil systems for understanding processes associated with allelopathic interaction: one example. In: Inderjit, Dakshini, K.M.M., Einhellig, F.A. (Eds.), Allelopathy: Organisms, Processes and Application. ACS Symposium Series 582. American Chemical Society, Washington, DC, USA, pp. 127–131. Bonanomi, G., Del Sorbo, G., Mazzoleni, S., Scala, F., 2007. Autotoxicity of decaying tomato residues affects susceptibility of tomato to Fusarium wilt. J. Plant Pathol. 89 (2), 219–226. Cakmak, I., Marschner, H., 1992. Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase in bean leaves. Plant Physiol. 98, 1222–1227. Cao, P.R., Luo, S.M., 1996. Studies of autotoxicity of tea tree. Tea Guangdong 2, 9–11. Chaudhary, N.Y., Khan, A.S., 2007. Role of mercury and exogenous IAA on xylem vessels and sieve elements in Cucumis sativus L. Pak. J. Bot. 39 (1), 135–140. Chen, C.T., Li, C.C., Kao, C.H., 1991. Senescence of rice leaves XXXI. Changes of chlorophyll, protein, and polyamine contents and ethylene production during senescence of a chlorophyll-deficient mutant. J. Plant Growth Reg. 10, 201–205. Chou, C.H., Lin, H.J., 1976. Autotoxication mechanism of Oryza sativa. I. Phytotoxic effects of decomposing rice residues in soil. J. Chem. Ecol. 2, 353–367. Crawford, N.M., Guo, F.Q., 2005. New insights into nitric oxide metabolism and regulatory functions. Trends Plant Sci. 10, 195–200. Cruz-Ortega, R., Alvarez-Anorve, M., Romero-Romero, M.T., Lara-Nunez, A., Anaya, A.L., 2008. Growth and oxidative damage effects of Sicyos deppei weed on tomato. Allelopathy J. 21 (1), 83–94. De Araújo, S.A.M., Silveira, J.A.G., Almeida, T.D., Rocha, I.M.A., Morais, D.L., Viéga, R.A., 2006. Salinity tolerance of halophyte Atriplex nummularia L. grown under increasing NaCl levels. Rev. Bras. Eng. Agric. Ambient. 10, 848–854. Egamberdieva, D., 2009. Alleviation of salt stress by plant growth regulators and IAA producing bacteria in wheat. Acta Physiol. Plant 31, 861–864.

Sunaina, N.B. Singh / Scientia Horticulturae 192 (2015) 211–217 Einhellig, F.A., 1995. Mechanism of action of allelochemicals in allelopathy. In: Inderjit, Dakshini, K.M.M., Einhellig, F.A. (Eds.), Allelopathy, Organisms, Processes and Applications, ACS Symposium Series, vol. 520. American Chemical Society, Washington, DC, pp. 96–116. Enping, Z., Shuhong, Z., Liangliang Li, 2015. Effect of tomato (Solanum lycopersicum L.) plant parts extracts, root exudates and tomato grown soil extract on seed germination and seedlings growth of tomato. Allelopathy J. 35 (1), 1–10. Farkhondeh, R., Nabizadeh, E., Jalilnezhad, N., 2012. Effect of salinity stress on proline content, membrane stability and water relations in two sugar beet cultivars. Int. J. Agri. Sci. 2, 385–392. Fazeli, F., Ghorbanli, M., Niknam, V., 2007. Effect of drought on biomass, protein content, lipid peroxidation and antioxidant enzymes in two Sesame cultivars. Biol. Plant 51, 98–103. Gangwar, S., Singh, V.P., Prasad, S.M., Maurya, J.N., 2011. Differential responses of pea seedlings to indole acetic acid under manganese toxicity. Acta Physiol. Plant 33, 451–462. Guru Devi, R., Pandiyarajan, V., Gurusaravanan, P., 2012. Alleviating effect of IAA on salt stressed Phaseolus mungo (L.) with reference to growth and biochemical characteristics. Recent Res. Sci. Technol. 4, 22–24. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts. 1. Kinetics and stoichiochemitry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189–198. Hedge, J.E., Hofreiter, B.T., 1962. Estimation of carbohydrate. In: Whistler, R.L., Be Miller, J.N. (Eds.), Methods in Carbohydrate Chemistry. Academic Press, New York, pp. 17–22. Hemeda, H.M., Klein, B.P., 1990. Effects of naturally occurring antioxidants on peroxidase activity of vegetable extracts. J. Food Sci. 55, 184–185. Hoagland, D.R., Arnon, D.I., 1950. The water culture method for growing plants without soil. In: California Agriculture Experiment Station. University of California, Circular, Berkeley (CA), pp. 347. Inderjit, Duke, S.O., 2003. Ecophysiological aspects of allelopathy. Planta 217, 529–539. Iqbal, M., Ashraf, M., 2007. Seed treatment with auxins modulates growth and ion partitioning in salt-stressed wheat plants. J. Integr. Plant Biol. 49, 1003–1015. Javid, M.G., Sorooshzadeh, A., Moradi, F., Sanavy, S.A.M.M., Allahdadi, I., 2011. The role of phytohormones in alleviating salt stress in crop plants. A.J.C.S. 5 (6), 726–734. Jaworski, E., 1971. Nitrate reductase assay in intact plant tissue. Biochem. Biophys. Res. 43, 1274–1279. Kaiser, W.M., Spill, D., Glaab, J., 1993. Rapid modulation of nitrate reductase in leaf and roots: indirect evidence for the involvement of protein phosphorylation/dephosphorylation. Physiol. Planta 89, 557–562. Kato, T., Takeda, K., 1993. Endogenous abscisic acid in developing grains on primary and secondary branches of rice (Oryza sativa L.). Jpn. J. Crop Sci. 62, 636–637. Kaya, C., Ashraf, M., Dikilitas, M., Tuna, A.L., 2013. Alleviation of salt stress-induced adverse effects on maize plants by exogenous application of indoleacetic acid (IAA) and inorganic nutrients – A field trial. Aust. J. Crop Sci. 7 (2), 249–254. Kaya, C., Kirnak, H., Higgs, D., 2001. Enhancement of growth and normal growth parameters by foliar application of potassium and phosphorus on tomato cultivars grown at high (NaCl) salinity. J. Plant Nutr. 24, 357–367. Kaya, C., Tuna, A.L., Okant, A.M., 2010. Effect of foliar applied kinetin and indole acetic acid on maize plants grown under saline conditions. Turk. J. Agr. For. 34, 529–538. Khan, M.A., Gul, B., Weber, D.J., 2004. Action of plant growth regulators and salinity on seed germination of Ceratoides lanata. Can. J. Bot. 82, 37–42. Kim, Y.S., Kil, B.S., 1989. Identification and growth inhibition of phytotoxic substances from tomato plants. Kor. J. Botany 32, 41–49. Lichtenthaler, H.K., 1987. Chlorophyll and carotenoids: pigments of photosynthetic bio-membranes. In: Packer, L., Douce, R. (Eds.), Methods in Enzymology. Academic Press, San Diego, pp. 350–382. Lin, W.X., Kim, K.U., Shin, D.H., 2000. Rice allelopathic potential and its modes of action on barnyard grass (Echinochloa crus-galli). Allelopathy J. 7, 215–224. Liu, Y., Wang, X., Zeng, G., Qu, D., Gu, J., Zhou, M., Chai, L., 2007. Cadmium-induced oxidative stress and response of the ascorbate glutathione cycle in Bachmeria nivea (L.). Gaud. Chemosphere 69, 99–107. Lowry, O.H., Rosebrough, N.J., Fan, A.L., Randall, R.I., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275. Mansour, M.M.F., 2012. Plasma membrane permeability as an indicator of salt tolerance in plants. Biol. Plant, http://dx.doi.org/10.1007/s10535-012-0144-9 Miller, D.A., 1996. Allelopathy in forage crop systems. Agronomy J. 88, 854–859. Mizutani, J., 1984. Allelochemicals in Tomato. In Survival of Mankind and Plant Production. In: Tamura, S., Takahashi, N. (Eds.). Tokyo Univ. Press, Tokyo, pp. 286–287 (in Japanese). Møller, I.M., Jensen, P.H., Hansson, A., 2007. Oxidative modifications to cellular components in plants. Annu. Rev. Plant Biol. 58, 459–481.

217

Naeem, M., Bhatti, I., Ahmad, R.H., Ashraf, M.Y., 2004. Effect of some growth hormones (GA3, IAA and Kinetin) on the morphology and early or delayed initiation of bud of lentil (Lens culinaris medik). Pak. J. Bot. 36 (4), 801–809. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880. Niu, X., Bressan, R.A.P., Hasegawa, M.J., Pardo, M., 1995. Ion homeostasis in NaCl stress environments. Plant Physiol. 109, 735–742. Nürnberger, T., Nennstiel, D., Jabs, T., Sacks, W.R., Hahlbrock, K., Scheel, D., 1994. High affinity binding of a fungal oligopeptide elicitor to parsley plasma membranes triggers multiple defence responses. Cell 78, 449–460. Rice, E.L., 1984. Allelopathy, 2nd ed. Academic Press, London. Ritenour, M.A., Sutter, E.G., William, D.M., Saltveit, M.E., 1996. IAA content and auxiliary bud development in relation to russet spotting in harvested Iceberg lettuce. J. Soc. Hortic. Sci. 121 (3), 543–547. Rubio, M.C., Gonzalez, E.M., Minchin, F.R., Webb, K.J., Arrese-Igor, C., Ramos, J., Becana, M., 2002. Effect of water stress on antioxidant enzymes of leaves and nodules of transgenic alfalfa over expressing superoxide dismutases. Physiol. Planta 115, 531–540. Ruiz, J.M., Sanchez, E., Garcia, P., Lopez-Lefebre, L.R., Rosa, R.M., Romero, L., 2002. Proline metabolism and NAD kinas activity in green bean plants subjected to cold-shock. Phytochemistry 59, 473–478. Sairam, R.K., Deshmukh, P.S., Saxena, D.C., 1998. Role of antioxidant systems in wheat genotypes tolerance to water stress. Biol. Planta 41, 387–394. Sannigrahi, A.K., Chakrabortty, S., 2005. Allelopathic effects of weeds on germination and seedling growth of tomato. Allelopathy J. 16 (2), 289–294. Sheltel, N.L., Balke, N.E., 1983. Plant growth response to several allelopathic chemicals. Weed Sci. 31, 293–298. Singh, K., Rathore, S., 1998. Seed and protein yield of mung in response to treatment with indole acetic acid (IAA). Plant Physiol. 32 (2), 133–137. Singh, N.B., Singh, A., Singh, D., 2008. Autotoxic effects of Lycopersicon esculentum. Allelopathy J. 22, 429–442. Singh, N.B., Singh, D., Singh, A., 2010. Allelochemicals enhance the sever effects of water stress in seedling of Phaseolus mungo. Allelopathy J. l25, 185–194. Synkova, H., Semoradova, S., Burketova, L., 2004. High content of endogenous cytokinins stimulates activity of enzymes and proteins involved in stress response in Nicotiana tabacum. Plant Cell Tissue Organ Cult. 79, 169–179. Thapar, R., Singh, N.B., 2005. Effect of Ocimum americanum leachates on seed germination and seedling growth of on germination, growth and metabolism of Parthenium hysterophorus L. Allelopathy J. 16, 301–308. Tognetti, V.B., Müllenbock, P.E.R., Van Breusegem, F., 2012. Stress homeostasis—the redox and auxin perspective. Plant Cell Environ. 35, 321–333. Torres-Garcia, J.R., Escalante-Estrada, J.A., Rodriguez-Gonzalez, M.T., Ramirez-Ayala, C., Martinez-Moreno, D., 2009. Exogenous application of growth regulators in snap bean under water and salinity stress. J. Stress Physiol. Biochem. 5 (3), 13–21. Ünyayar, S., Keles¸, Y., C¸ekic¸, F.Ö., 2005. The antioxidative response of two tomato species with different drought tolerances as a result of drought and cadmium stress combinations. Plant Soil Environ. 51, 57–64. Vaughan, D., Ord, B.G., 1991. Extraction of potential allelochemicals and their effects on root morphology and nutrient content. In: Atkinson, D. (Ed.), Plant Root Growth, an Ecological Perspective. Blackwell Scientific, London, pp. 399–421. Wang, Y., Mopper, S., Hasentein, K.H., 2001. Effects of salinity on endogenous ABA, IAA, JA, and SA in Iris hexagona. J. Chem. Ecol. 27, 327–342. Wu, F.Z., Liu, D., Wang, D.K., Laun, F.S., Wang, W., Kong, X.M., 1997. The effects of different years of continuous cropping on the vitality of root system and their qualities in the plastic house tomatoes. J. North Agric. Univ. 28, 33–38. Yadav, K., Singh, N.B., 2013. Effects of benzoic acid and cadmium toxicity on wheat seedlings. Chil. J. Agric. Res. 73 (2), 168–174. Yu, J.Q., Lee, K.S., Matsui, Y., 1993. Effect of additional of activated charcoal to the nutrient solution on the growth of tomato in hydroponic culture. Soil Sci. Plant Nutr. 39, 13–22. Yu, J.Q., Matsui, Y., 1997. Effect of root exudates of cucumber (Cucumis sativus) and allelochemicals on uptake by cucumber seedlings. J. Chem. Ecol. 23, 817–827. Zhang, E.P., Zhang, S.H., Li, W.Z.L.L., Li, T.L., 2010. Effects of exogenic benzoic acid and cinnamic acid on the root oxidative damage of tomato seedlings. J. Hortic. For. 2 (2), 22–29. Zheng, J.H., Ye, S.F., Yu, J.Q., 2004. Causes of soil sickness in cropping of vegetable crops and its biological control. China Veg. 3, 56–139. Zhou, Z.H., Luo, S.M., Mou, Z.P., 1997. Allelopathic effects of tomato. Chin. J. Appl. Ecol. 8, 445–449. Zhou, Z.H., Luo, S.M., Mou, Z.P., 1998. Study on allelopathic potential of tomato (Lycopersicon). J. South China Agric. Univ. 19, 56–60.